1. Field of the Invention
The present invention relates generally to the circadian regulation of the sleep/wake cycle, and more specifically to methods of identifying agents that affect calcium activated potassium channel activity and thereby modulate the sleep/wake cycle, and to methods of modulating the sleep/wake cycle by affecting calcium activated potassium channel activity.
2. Background Information
Most organisms undergo a rhythmic pattern of sleep and wakefulness that cycles over a twenty-four hour period and generally is linked to the day/night cycle. In human adults, for example, sleep onset generally begins about four to five hours after nightfall, and spontaneous awakening occurs about one to two hours after sunrise (see Young and Kay, Nat. Rev. Genet. 2:702, 2001). Many individuals, however, suffer from abnormalities in the normal sleep/wake cycle, including, for examples, individuals that suffer from insomnia and individuals that suffer from disorders such as narcolepsy, in which sleep onset can occur at any time of day, or familial advanced sleep phase syndrome (FASPS), in which the time of sleep onset and awakening occur earlier than normal (see, for example, Young and Kay, supra, 2001).
A genetic mutation was recently linked to FASPS in humans, and is the first example of a genetic defect associated with a defect in the sleep/wake cycle in humans (Toh et al., Science 291:1040, 2001). The identification of such a mutated gene, and its normal counterpart, provide a means to develop assays for identifying drugs that correct, or at least decrease, the effect due to the mutation, thus allowing an individual with the mutation to have a more normal pattern of sleep and wakefulness. Although drugs that ameliorate the effect due to the mutation in FASPS will be a great help to individuals having that mutation, the drugs are not likely to be useful for individuals suffering from other sleep disorders such as insomnia. As such, the majority of individuals suffering from disorders of the sleep/wake cycle such as insomnia must continue to rely on relatively non-specific drugs, including prescription drugs such as benzodiazepine agonists and over-the-counter drugs, which often contain antihistamines. Such drugs, however, act generally and, while they can assist in helping a person sleep, they also can have undesirable side effects, including causing confusion and loss of balance.
Clearly, it would be preferable to identify additional specific genes involved in regulating the sleep/wake cycle so that individual suffering from sleep disorders so that the gene products could be used as targets in screening assays to identify drugs useful for specifically modulating the sleep/wake cycle. Microarray technology provides a means to identify the expression of a large number of genes in a single assay and, therefore, provides a powerful tool for identifying genes of interest, including those involved in regulating the sleep/wake cycle. A microarray is formed by linking a large number of discrete polynucleotide sequences, for example, a population of polynucleotides representative of a genome of an organism, to a solid support such as a microchip, glass slide, or the like, in a defined pattern. By contacting the microarray with a nucleic acid sample obtained from a cell of interest, and detecting those polynucleotides expressed in the cell that hybridize specifically to complementary sequences on the chip, the pattern formed by the hybridizing polynucleotides allows the identification of clusters of genes that are expressed in the cell. Furthermore, when each polynucleotide linked to the solid support is known, the identity of the hybridizing sequences from the nucleic acid sample can be identified.
Microarray technology provides a means to identify coordinate gene expression simply by comparing patterns of hybridization. For example, by comparing the hybridization pattern of nucleic acid molecules obtained from cells of an individual suffering from a disease with the nucleic acids obtained from the corresponding cells of a healthy individual, clusters of genes that are differentially expressed can be identified. The identification of such differentially expressed genes provides a means to identify new genes, and provides insight as to the pattern of gene expression that occurs in a normal organism or in an organism suffering from a pathologic condition.
Microarray technology further allows the identification of clusters of genes that are coordinately regulated and that encode proteins common to particular intracellular pathways. Thus, microarray technology has been used to determine that proteins involved in metabolic pathways such as photosynthesis in plants and cuticle formation and lipid metabolism in fruit flies are encoded by genes that are coordinately regulated and, further, that such coordinate expression is circadian regulated, i.e., cycles with approximately twenty-four hour periodicity corresponding to day and night.
Despite the large number of genes that have been identified as circadian regulated, the key gene or genes that determine the sleep/wake cycle in humans have remained elusive. The identification of such a gene or genes would provide a target for drugs that could be used, for example, to specifically induce sleep in individuals suffering from insomnia, without causing undesirable side effects common to currently used drugs. Thus, a need exists to identify genes that encode proteins that regulate the sleep/wake cycle. The present invention satisfies this need and provides additional advantages.